Table 1.

Comparison of SCRaMbLE to common S. cerevisisae strain improvement strategies

Technique	SCRaMbLE	Mass mating/genome shuffling	Transcription machinery engineering/rewiring	Deletion library screening	Targeted deletion/overexpression/underexpression	Random mutagenesis/directed evolution
Strain prerequisites	Synthetic chromosomes and recombinase vectors	Natural isolates with desired properties	Efficient transformation into strain	Deletion library, efficient transformation into strain	—	—
Prior knowledge	—	Phenotypic analysis of strains	Identification of transcriptional regulators/regulated promoters	—	Prior gene characterisation studies, knowledge of metabolic processes	—
Preparation work	Transform pathway into one strain	Transform pathway into half of mating strains	Large-scale library construction	Transform pathway into many strains	Construction of TAR cassettes	Transform pathway into one strain
Technical requirements	Basic strain cultivation	Strain mating/protoplast fusion, tetrad dissection	DNA assembly, library scale DNA transformation	Library scale DNA transformation	DNA assembly/gene editing tools	Strain cultivation, chemostat/tubidostat, mutagenesis
Timescale to improved strain	1–2 weeks	Months to years	Months to years	Weeks to months	Months	Months to years
Strain diversity generated	Coding sequence deletions, inversions, duplications, translocations	Allele combinations, polyploidy	Novel regulation	Coding sequence deletions	Specific targeted changes	Point mutations
Other factors	—	High prevalence of deleterious alleles, mitotic instability	High throughput screening often necessary	Higher numbers of combined knockouts exponentially increases transformation requirements	Understanding of biological mechanisms often not sufficient for effective improvement	High number of off-target effects, selective pressure often required

Technical requirements do not include screening methods, which are common to all methods. Timescale to improved strain estimates length of time from introduction of a heterologous pathway to isolation and characterisation of a strain with an improved phenotype